Proteomic Analysis of NF90 (Family) Complexes

描述（由申请人提供）：双链RNA结合蛋白NF90家族是通过ILF3基因转录本的选择性剪接产生的。该家族的成员在几个不同的亚细胞位置被发现，并与各种蛋白质复合物相互作用，提示在各种细胞过程中发挥功能，包括基因表达、DNA复制和修复、细胞生长和抗病毒免疫反应。虽然至少有7种独特的蛋白质产物已经从cdna中预测出来，但已经确定在人类细胞系中有四个家族成员占主导地位，并且这些同工异构体在瞬时表达试验中表现出不同水平的活性。这些观察结果提出了一种可能性，即同种异构体参与在细胞内显示不同功能的特定复合物。为了解决这一假设，将从最近开发的，具有良好特征的表达四种亚型的稳定细胞系中分离出含有天然NF90的复合物。这些复合物的组成将利用蛋白质组学技术确定。一旦已知非同步人类细胞的复合物组成（目的1），将使用类似的方法来研究含有不同NF90成员的复合物的动力学作为细胞周期期和病毒感染的函数（目的2）。

DESCRIPTION (provided by applicant): The NF90 family of double-stranded RNA binding proteins is generated by alternative splicing of transcripts from the ILF3 gene. Members of this family have been found in several different subcellular locations and interact with varied protein complexes, suggestive of functions in various cellular processes including gene expression, DNA replication and repair, cell growth, and the anti-viral immune response. Although at least 7 unique protein products have been predicted from the cDNAs, it has been established that four family members predominate in human cell lines and that these isoforms exhibit different levels of activity in transient expression assays. These observations raise the possibility that the isoforms participate in specific complexes that display distinct functions within cells. To address this hypothesis, native NF90 containing complexes will be isolated from recently developed, well-characterized stable cell-lines expressing the four isoforms. The composition of the complexes will be established using proteomic techniques. Once the complex compositions are known for asynchronous human cells (Aim 1), a similar approach will be used to investigate the dynamics of the complexes containing different NF90 members as a function of cell cycle phase and viral infection (Aim 2).